Telomeres are repeated hexanucleotide sequences at the ends of linear chromosomes, which serve to protect them from recognition as chromosomal breaks. Asymmetric replication of DNA would lead inevitably to a loss of genetic material, and the telomere repair molecular machinery (a reverse transcriptase, RNA template, and associated proteins) functions to maintain genomic integrity. Telomerase deficiency manifests with short telomeres. Mutations in DKC1 and in TERC (the RNA template subunit of the complex) are etiologic in dyskeratosis congenita, a constitutional form of aplastic anemia. Mutations in TERT (encoding telomerase, the rate limiting enzymatic component of the complex) and in TERC occur in apparently acquired aplastic anemia and other diseases. Male hormones, long used to treat aplastic anemia, act to regulate TERT transcription and telomerase activity. While critical telomere shortening leads to either cell senescence or apoptosis, occasionally cells become aneuploid due to end-to-end fusion of chromosomes. Thus, telomere attrition is a mechanism for oncogenesis, in which chromosome instability rather than the cumulative acquisition of somatic mutations in specific genes is etiologic. In the clinic, we routinely measure telomere length commercially by a CLIA flow-FISH method, and in our research laboratory by gene amplification qpcr. Measurement of telomere length in clinical samples is required for the adequate diagnosis of aplastic anemia. Telomere length and the rate of loss of telomere are predictive of late events after treatment with immunosuppression, and in other clinical circumstances. Our research laboratory is proficient in molecular assays related to telomere maintenance and function.Having successfully completed our clinical protocol testing danazol at high doses and prolonged administration for clinical efficacy and telomere effects in patients with telomere disease, we have initiated a new, low dose danazol trial. In the current, patients are randomized to initially receive either half or quarter doses (400 mg or 200 mg daily) compared to the original protocol, for 6 months, and then crossed over the other dose for a further 6 months. These regimens should avoid the toxicities of high dose sex hormones. The design of the current protocol also addresses deficiencies of the original study: 1. We will utilize both q-pcr and also flow-FISH for telomere length and 2. 6 month observation and wash-out periods precede and follow drug administration in order to provide baseline telomere attrition information. Additionally, because of the suggestion of stabilization and possibly improvement in pulmonary function in the earlier protocol, the inclusion criteria have been expanded to recruit patients with mainly lung manifestions of telomeropathy. Accrual is proceeding as anticipated to the low dose danazol protocol. To date, eight patients have been enrolled on study. Also, in the clinic, relating to telomere disease especially but relevant to the larger clinical issue of distinction between constitutional and acquired etiologies for bone marrow failure, we systematically screen by genomics, in collaboration with the University of Chicago, patients presenting to our clinic with a wide variety of manifestations of bone marrow failure. We assess for mutations and polymorphisms in >50 genes etiologic in inherited marrow failure syndromes for both research purposes and clinical reporting to the patient. For comparative purposes, we also have data from collaborators at a marrow failure center in Sao Paolo, Brazil. To date, our results indicate: first, patients with acute onset of severe aplastic anemia, defined by convention based on peripheral blood counts, very rarely have mutations in constitutional marrow failure genes, and second, that clinical correlates such as family history and multi-organ involvement are highly predictive of a germline etiology and positive genomic testing. We are applying machine learning techniques to develop an algorithm for specialists to allow distinction between acquired and inherited marrow failure syndromes. We utilize our referral base to investigate novel genes in individual patients and their families. In collaboration with treating physicians in Lebanon, we have characterized a remarkable large, consanguineous pedigree with homozygous RTEL1 mutations that present with an extraordinary range of clinical phenotypes, from fatal myelodysplastic syndrome and leukemia early in life to mild evidence of marrow failure in adults. In an Iranian family with multiple members suffering thrombocytopenia, we identified a mutation in a highly conserved region of the SBF2 gene. SBF2 previously has only been found mutated in Charcot-Marie-Tooth syndrome. Absent a functional assay, we are introducing this mutation into a megakaryocytic cell line and primary CD34 cells in order to characterize a molecular defect in platelet formation and potentially a novel pathway in megakaryocytopoiesis. As a component of our single cell effort, we are exploring scRNAseq in constitutional marrow failure syndromes. In the telomeropathies and related diseases, numbers of CD34 marrow cells are inadequate for such efforts, We have been successful in defining the hematopoietic defect in GATA2 deficiency, as described in our complementary annual report. DADA2 or deficiency of adenosine deaminase-2 presents with a wide range of clinical symptoms and signs, including marrow failure, autoimmune manifestations, and vasculitis and thrombosis. In this disease, the macrophage has been the subject of most interest, and we have applied RNA sequencing of single cells to this population. We could distinguish among macrophage subsets by imputation of sequence and found a marked increase in the nonclassical subtype; among all subtypes there was striking upregulation of immuen pathway genes, especially for interferon signaling, and distinctive upregulation of the NF-kappaB in the nonclassical cells. These experiments provide novel insights into the pathogenesis of this syndrome and potentially the basis for a diagnostic test. Last, because telomeropathy is associated with chromosome instability and myeloid neoplasm--and represents an alternative pathway to acquisition of mutations in the development of cancer--we are testing new methods for sensitive detection of single copy number variation as a harbinger of gross aneuploidy and transformation.
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